BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*

Objective To detect the association between rs4646999 polymorphisms in the promoter region of the c-Jun and the prognosis of sporadic colorectal cancer. Methods rs4646999-673C>T genetypes were deter-mined by Taqman-MGB probes in 436 colorectal cancer cases. The survival curve was analyzed by Kaplan-Meier analysis and Cox regression.Western blot was used to analyze the expression levels of c-Jun protein in different gen-otypes. Results Univariate analysis showed that the cumulative survival rate of patients with rs4646999TT geno-type was significantly higher than that of patients with CT and CC genotype. Multivariate Cox regression analysis showed that the differentiation,lymph node metastasis,distant metastasis,TNM stage and rs4646999 genetypes were prognostic factors.Compared with the carriers of TT genotype,CT/CC complex genotypes were associated with poor prognosis of colorectal cancer(P<0.05).Protein expression analysis showed that the expression of c-Jun pro-tein in CC genotype was increased.In contrast,the TT genotype was decreased.Conclusions This study provided the evidence that rs4646999-673C>T genetic variant in c-Jun promoter regions is associated with the poor survival prognosis of colorectal cancer,possibly by elevating the protein expression levels that appeared to up-regulate activ-ity of c-Jun thus tumorigenesis.

Objective: To investigate the effect of irradiation to anastomosis from preoperative radiotherapy for patients with rectal cancer by studying the pathological changes. Methods: In this retrospective study, patients enrolled in the FOWARC study from January 2011 to July 2014 in the Sixth Affiliated Hospital of Sun Yat-Sen University were included. In the FOWARC study, enrolled patients with local advanced rectal cancer were randomly assigned to receive either neoadjuvant chemo-radiotherapy or chemotherapy. Among these patients, 23 patients were selected as radiation proctitis (RP)group, who fulfilled these conditions: (1) received neoadjuvant chemo-radiotherapy followed by sphincter-preserving surgery; (2) developed radiation proctitis as confirmed by preoperative imaging diagnosis; (3) had intact clinical samples of surgical margins. Twenty-three patients who had received neoadjuvant chemo-radiotherapy but without development of radiation proctitis were selected as non-radiation proctitis (nRP) group. Meanwhile, 23 patients received neoadjuvant chemotherapy only were selected as neoadjuvant chemotherapy (CT) group. Both nRP and CT cases were selected by ensuring the basic characteristics such as sex, age, tumor site, lengths of proximal margin and distal margin all maximally matched to the RP group. Both proximal and distal margins were collected for further analysis for all selected cases. Microscopy slices were prepared for hematoxylin & eosin staining and Masson staining to show general pathological changes, and also for immunohistochemistry with anti-CD-34 as primary antibody to reveal the microvessel. Microvessel counting in submucosal layer and proportion of macrovessel with stenosis were used to evaluate the blood supply of the proximal and distal end of anastomosis. A modified semi-quantitative grading approach was used to evaluate the severity of radiation-induced injury. Either ANOVA analysis, Kruskal-Wallis rank-sum test or χ² test was used for comparison among three groups, and Mann-Whitney U test was used for comparison between two groups. Results: Compared to group of neoadjuvant chemotherapy only, patients receiving neoadjuvant chemo-radiotherapy had lower microvessel count in both proximal and distal margins (M(Q_R): proximal, 25.5 (19.6) vs. 50.0 (25.0), Z=3.915, P=0.000; distal, 20.5 (17.5) vs. 49.0 (28.0), Z=3.558, P=0.000), higher proportions of macrovessel with stenosis (proximal, 9.5% (23.8%) vs. 0, Z=3.993, P=0.000; distal, 11.5%(37.3%) vs. 0 (2.0%), Z=2.893, P=0.004), higher histopathologic score (proximal, 4.0 (2.0) vs. 1.0 (2.0), Z=6.123, P=0.000; distal, 5.0 (3.0) vs. 2.0 (1.0), Z=4.849, P=0.000). In patients receiving neoadjuvant chemo-radiotherapy, compared to nRP group, RP group had lower microvessel count in both proximal and distal margins (proximal, 19.0 (23.0) vs. 30.4 (38.0), Z=2.845, P=0.004; distal, 19.0 (13.0) vs. 30.0(29.1), Z=2.022, P=0.043), higher proportions of macrovessel with stenosis (proximal, 23.0% (40.0%) vs. 0(11.0%), Z=3.248, P=0.001; distal, 27.0% (45.0%) vs. 3.0% (19.0%), Z=2.164, P=0.030). Rate of anastomotic leakage for CT, nRP and RP group were 8.7% (2/23), 30.4% (7/23), and 52.2% (12/23), and the differences among three groups were statistically significant (χ²=10.268, P=0.007). Conclusion Radiation-induced injury existed on both margins of the resected rectal site after preoperative radiotherapy, and those diagnosed as radiation proctitis had more severe microvascular injury.

Objective To investigate the expressions of PTEN and MDM2 in bladder transitional cell carcinoma (BTCC) and their clinical significance.Methods The expressions of PTEN and MDM2 were detected by tissue immunohistochemistry test (SP method) in BTCC (n =80) and normal bladder tissues (n =20).The relationship between PTEN and MDM2 as well as their correlations with clinical pathological features were analyzed.Results The positive rate of PTEN in different pathological grading (G1,G2,G3)and clinical staging [superficial (Tis ～ T1),infiltration (T2 ～ T4)] was (86.20％,74.07％,37.50％ ;80.00％,46.67％),respectively,with a significant difference (x2 =15.004,P ＜ 0.01 ; x2 =9.497,P ＜0.01).The positive rate of MDM2 in different pathological grading(G1,G2,G3) and clinical staging [superficial (Tis ～ T1),infiltration (T2 ～ T4)] was (82.75％,55.55％,37.50％ ; 70.00％,43.35％),respectively,with a significant differcnce(x2 =11.543,P ＜ 0.01 ; x2 =5.556,P ＜ 0.05).The expression of PTEN was negatively correlated with that of MDM2 in BTCC (r =-0.611,P ＜ 0.05).Conclusions Expressions of PTEN and MDM2 might be involved in the BTCC pathogenesis.The combined detection of PTEN and MDM2 might be of great value in the prediction of tumor behavior and prognosis.